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Abstract

Protease activated receptor (PAR) stimulation induces procoagulant phenotypes on platelets leading to thrombin generation (TG); however, the efficacy of PAR1 versus PAR4 in these processes has not been compared. We developed a novel TG assay to monitor platelet assembly of individual coagulation complexes (prothrombinase, extrinsic Xase, intrinsic Xase). Conditions were established in which certain cofactors were withheld and no thrombin is generated without platelet stimulation and provision of the coagulation complex cofactor. TG targeting the prothrombinase complex assembly in which factor V (FV) was withheld (FII+FXa+ATIII) lead to roughly double the peak thrombin and shorter lagtimes for PAR4 versus PAR1 stimulation. In accordance with these findings, dose response curves of PAR1-AP and PAR4-AP mediated secretion and binding of FV on the cell surface using flow cytometry (FC) revealed a maximal response for PAR4 double that of PAR1. TG experiments targeting intrinsic Xase assembly (FII+FX+FIXa+ATIII) were twice as high as isolated prothrombinase conditions highlighting the ability of the intrinsic Xase complex to amplify TG. These results also demonstrated that platelets secrete and bind functional factor VIII (FVIII). Once again PAR4 mediated responses including FVIII secretion and binding, and TG exceeded PAR1 by two fold. Finally, we observed a rapid (within 5 min) mobilization of tissue (TF) to the cell surface following PAR stimulation by FC. TG targeting extrinsic Xase assembly (FII+FX+FVIIa+TFPI+ATIII) was able to generate thrombin in response to stimulation yet levels were 5 and 10 times lower than prothrombinase and intrinsic Xase conditions, respectively. In conclusion, we have shown that platelets harbor FV, FVIII and TF in internal stores that can be utilized to generate thrombin on the surface of the platelet and that PAR4 stimulation induces the provision of more FV and FVIII, consistently yielding more thrombin than PAR1 stimulation. These data have important clinical impact in light of recent efforts to develop thrombin receptor antagonists as agents to treat thrombotic disorders, suggesting that perhaps a PAR4 antagonist would be a more effective strategy to control pathologic thrombosis driven by platelet TG.